培養(yǎng)基的制備步驟詳情說明
發(fā)布時間：
2022-12-27
作者：
不同類型的培養(yǎng)基制備程序也不同，主要可分為：配料、溶化、矯正pH、澄清過濾、分裝、滅菌及檢定8個步驟。
1，配料：按培養(yǎng)基處方準確稱取各種成分，先在三角燒瓶中加入少量蒸餾水，再加入各種成分，以防蛋白胨等粘附于瓶底，然后再以剩余的水沖洗瓶壁。
2，溶化：將各種成分混勻于水中，最好以流通蒸氣溶化半小時，如在電爐上溶化應(yīng)隨時攪拌，如有瓊脂成分時更應(yīng)注意防止外溢。溶化完畢，補足失去的水分。
3，矯正pH：pH測定，取與標準管同口徑的試管（通常用華氏試管）3支，于第1、3管各加入欲測定pH的的培養(yǎng)基5ml，并于第一管中加入0.2g／L的酚紅0.25ml作為測定管，混勻醫(yī)學(xué)|教育網(wǎng)搜集整理；于第2管加入蒸餾水5ml，第4管為pH標準比色管。 pH的校正，若測定管過酸或過堿可用0.1mol／L氫氧化鈉或0.1mol／L鹽酸溶液矯正，直至顏色與標準管相同為止，加堿或加酸時要精確緩慢，每加1滴后要充分混勻，比色后再加第2滴（有時僅加半滴）準確記錄加入的量。 計算，設(shè)5ml培養(yǎng)基矯正pH至7.4時需0.1mol／L氫氧化鈉0.15ml，現(xiàn)有培養(yǎng)基4990ml，需加氫氧化鈉的量可按下列方法計算：5∶4990＝0.15∶X X＝0.15×4990/5＝149.7（ml），如將此0.1mol／L的氫氧化鈉改用1mol／L的氫氧化鈉時，則需14.9ml即可。
4，過濾澄清：培養(yǎng)基配制后一般都有沉渣或混濁出現(xiàn)，需過濾成清晰透明后方可使用，常用的過濾方法如下：
液體培養(yǎng)基必須清晰，以便觀察細菌的生長情況，常用濾紙過濾，亦可在加熱前加入用水稀釋的雞蛋白（1000ml培養(yǎng)基用1個雞蛋白）在100℃加熱后保持60～70℃40～60分鐘，使其不溶性物質(zhì)附于凝固的蛋白上而沉淀，然后再用虹吸法吸出上清液或以濾紙過濾。固體培養(yǎng)基如系瓊脂培養(yǎng)基，于加熱融化后需趁熱以絨布或兩層紗布中夾脫脂棉過濾；亦可用
自然沉淀法，即將瓊脂培養(yǎng)基盛人鋁鍋或廣口搪瓷容器內(nèi)，以高壓（103.43kPa）蒸汽融化15分鐘后，靜置高壓鍋內(nèi)過夜，次日將瓊脂傾出，用刀將底部沉渣切去，再融化即可收清晰的瓊脂培養(yǎng)基。
5，分裝：根據(jù)需要將培養(yǎng)基分裝于不同容量的三角燒瓶、試管中。分裝的量不宜超過容器的2／3以免滅菌時外溢。瓊脂斜面分裝量為試管容量的1／5，滅菌后須趁熱放置成斜面，斜面長約為試管長的2／3。半固體培養(yǎng)基分裝量約為試管長的1／3，滅菌后直立凝固待用。高層瓊脂分裝量約為試管的1／3，滅菌后趁熱直立，待冷后凝固待用。液體培養(yǎng)基分裝于試管中，約是試管長度的1／3。瓊脂平板：將滅菌（或加熱融化）后的培養(yǎng)基冷至50℃左右，以無菌手續(xù)傾人滅菌平皿內(nèi)，內(nèi)徑9cm的平皿傾注培養(yǎng)基約13～15ml，輕搖平皿底醫(yī)學(xué)|教育網(wǎng)搜集整理，使培養(yǎng)基平鋪于平皿底部，待凝固后即成，傾注時，切勿將皿蓋全部啟開，以免空氣中塵埃及細菌落入。新制成的平板培養(yǎng)基（簡稱平板），表面水分較多，不利于細菌的分離，通常應(yīng)將平皿倒扣擱置于37℃培養(yǎng)箱內(nèi)約30分鐘待平板平面干燥后使用。
6，滅菌：不同成分、性質(zhì)的培養(yǎng)基，可采用不同的滅菌方法。高壓蒸汽滅菌法：高壓滅菌的溫度與時間隨種類及數(shù)量的不同有所差別，一般少量分裝時高壓（103.43kPa）滅菌15分鐘即可，分裝量較大時，可高壓（103.43kPa）滅菌30分鐘，含糖的培養(yǎng)基高壓（55.16kPa）滅菌15分鐘。以免糖類被破壞。
7，檢定：每批制成后須經(jīng)檢定方可使用，檢定時將培養(yǎng)基放37℃溫箱內(nèi)培養(yǎng)24小時后，證明無菌，同時用已知菌種檢查在此培養(yǎng)基上生長繁殖及生化反應(yīng)情況，符合要求者方可使用。
8，保存：制好的培養(yǎng)基，不宜保存過久，以少量勤做為宜。每批應(yīng)注明名稱，分裝量，制作日期等，放在4℃冰箱內(nèi)備用。
Detailed description of the preparation steps of the medium
Different types of media have different preparation procedures. They can be divided into 8 steps: batching, melting, correcting pH, clarifying filtration, packing, sterilizing and testing.
1, ingredients: a variety of ingredients are accurately called according to the prescription of the medium. First, a small amount of distilled water is added to the triangular flask, and then a variety of ingredients are added to prevent peptone from sticking to the bottom of the bottle and then rinse the bottle wall with the remaining water.
2, dissolve: mix all ingredients in water, it is better to melt steam for half an hour, such as melting in the electric furnace should be stirred at any time, if there is agar composition, more attention should be paid to prevent spillover. After dissolving, the loss of water is made up.
3, correct pH:pH determination, take the test tube of the same caliber with the standard tube (usually with the Fahrenheit test tube) 3, the first, third tube each added pH culture medium 5ml, and add the 0.2g / L phenolic red 0.25ml as the measuring tube in the first tube, and mix the medical education network to collect and collate; the second tubes are added to the distilled water 5ml and the fourth tube is the pH standard. A colorimetric tube. PH correction, if the determination of tube peric acid or overalkali can be corrected by 0.1mol / L sodium hydroxide or 0.1mol / L hydrochloric acid solution, until the same color and the standard tube, adding alkali or adding acid is accurate and slow, after adding 1 drops, we should mix well, and then add second drops (sometimes only half a drop) after the color. It is calculated that 0.1mol / L sodium hydroxide 0.15ml is required to correct pH to 7.4 by 5ml medium. The existing medium 4990ml can be calculated by adding sodium hydroxide as follows: 5: 4990 = 0.15: X X = 0.15 x 4990/5 = 149.7 (ML).
4, filtration and clarification: after the media are prepared, sediment or turbidity usually appears and needs to be filtered for clarity and transparency.
The liquid medium must be clear in order to observe the growth of bacteria, filter paper often, and add water diluted chicken protein before heating (1000ml medium with 1 chicken proteins) at 100 C to maintain 60~70 centigrade for 40~60 minutes to precipitate its insoluble substance on the solidified protein and then siphon. The method sucked out the supernatant or filtered it with filter paper. Solid medium, such as agar medium, is heated and melted. It should be heated by flannelette or two layer gauze.
Natural precipitation method, forthcoming agar medium filled with aluminum pot or porcelain enamel container, with high pressure (103.43kPa) steam melting for 15 minutes, static high pressure pot overnight, the next day the agar out, cut the bottom of the bottom slag, then melt the agar medium.
5, sub packing: according to the need, the medium is divided into different capacity triangular flasks and test tubes. The amount of packing should not exceed 2 / 3 of the container to avoid spillover during sterilization. The amount of agar slant is 1 / 5 of the test tube capacity. After sterilization, it must be placed on the slope of heat and the length of the slope is about 2 / 3 of the length of the tube. The semisolid medium was packed at about 1 / 3 of the length of the tube, and was used for upright coagulation after sterilization. The level of agar in high level is about 1 / 3 of the test tube. After sterilization, it will be hot and upright. The liquid medium was packed in a test tube, about 1 / 3 of the length of the tube. The agar plate: the culture medium after sterilization (or heating melting) is cold to about 50 degrees C, in the sterilized flat dish with aseptic formalities, the plate of inner diameter 9cm is poured about 13 ~ 15ml, and the culture foundation is padded at the bottom of the plate. All dishes cover should be opened to prevent dust and bacteria from falling into the air. The newly made plate culture medium (flat plate) has more surface moisture and is not conducive to the separation of bacteria. Usually, the flat plate should be put on the plate for about 30 minutes in the incubator of 37 C to be used after the flat plane is dried.
6, sterilization: different ingredients and properties of the medium can be used with different sterilization methods. High pressure steam sterilization: the temperature and time of high pressure sterilization vary with the variety and quantity of the medium. High pressure (103.43kPa) can be sterilized for 15 minutes when a small amount of medium is divided, and when the medium of medium is large, the high pressure (103.43kPa) can be sterilized for 30 minutes, and the medium of sugar containing medium pressure (55.16kPa) is sterilized 15. Minute. Lest the sugar is destroyed.
7, verification: each batch of culture medium must be used by the calibrated side. After the test, the culture base is placed in the incubator of 37 C for 24 hours, and the asepsis is proved. At the same time, the growth propagation and biochemical reaction on this medium are checked with the known strains, which can be used in accordance with the requirements.
8, preservation: the medium should not be preserved for too long, and it should be done with a small amount of diligence. Each batch should specify the name, the amount of packing, the date of production, etc., and reserve it in the refrigerator at 4 degrees.